Toward a General Dehydrogenase Enzymatic Scaffold for Industrial Biocatalysis
By: Elliot T. Campbell
Publisher: [publisher not identified]
Published: 2011
Place of publication: [New York, N.Y.?]
Language: eng
Description: Enzymes catalyze a wide range of reactions with high efficiency and exquisite specificity. As such, they lend themselves well for use in a myriad of applications from the production of fine chemicals to use in biofuel cells. Demand for enzymes with novel specificities has risen in recent years, as they are "green" catalysts and may find use as environmentally friendly replacements for conventional catalysts in a variety of chemical processes. However, their widespread use has been hindered by a number of challenges, including high cost, low stability, and the requirement of expensive cofactors for catalysis. A significant amount of research has been done to address these limitations, but the approaches taken are rarely general, and thus it remains difficult to engineer industrially compatible enzymes. The ideal enzyme for use in these systems would be inexpensive to express and purify, extremely stable, easy to immobilize without loss of activity, able to use cheap, non-natural cofactors with improved stabilities and redox properties, and be rapidly evolvable for desired substrate specificities and reactions.

Here, we present a novel approach to satisfy these requirements. We begin with a designed enzyme scaffold with beneficial properties for use in these systems, and then engineer in cofactor and substrate specificity as required for the application. A thermostable alcohol dehydrogenase, AdhD, from the hyperthermophilic archaea Pyrococcus furiosus was selected as the scaffold for this work, as it possesses several features which make it an attractive candidate for protein engineering and downstream industrial applications. It can be expressed recombinantly in Escherichia coli in high yield, and is readily purified due to its extreme thermostability (half-life of 130 min at 100°C). Additionally, a thermostable scaffold will increase enzyme lifetimes in industrial applications, and provide resistance to chemical and thermal inactivation. AdhD belongs to the aldo-keto reductase superfamily, a large and diverse family of oxidoreductase enzymes, and shares the canonical (α/β)8-barrel fold and nicotinamide cofactor binding pocket.

AdhD has a strong preference for NAD(H) over NADP(H), and is active with a broad range of substrates. Lastly, the enzyme is monomeric, with no metal centers or disulfides, further simplifying engineering efforts. We began by examining cofactor binding in the AdhD enzyme through several rational mutations to the cofactor binding pocket. Guided by previous work examining cofactor specificity in the aldo-keto reductase superfamily, we identified two mutations, K249G and H255R, which had a significant impact on cofactor binding and activity. While characterizing the cofactor specificity double mutant, we discovered that the mutations also enabled the enzyme to utilize a truncated nicotinamide cofactor for catalysis. The benefit of improved cofactor diffusion was demonstrated through the creation of an enzymatic biofuel cell for the oxidation of D-arabinose. Next we examined the substrate specificity of the enzyme, utilizing a rational loop-swapping approach. AdhD was readily imparted with aldose reductase activity through the grafting of substrate binding loops from another AKR, human aldose reductase.

The chimeric loop mutants also retained activity with the model substrate for AdhD, but exhibited a complete reversal of cofactor specificity. Finally, we discuss the design and preliminary results of a novel selection step for the directed evolution of substrate specificity and catalytic activity. Taken together, this work describes the development of a general dehydrogenase enzymatic platform that can be adapted for use in a wide range of applications.
Notes: Department: Chemical Engineering.

Thesis advisor: Scott A. Banta.

Thesis (Ph.D.)--Columbia University, 2011.